The present invention relates to a crystal structure, and more particularly to the crystal structure of glutaminyl cyclase (QC).
The formation of N-terminal pGlu is an important posttranslational or co-translational event in the processing of numerous bioactive neuropeptides, hormones, and cytokines during their maturation in the secretory pathway. The N-terminal pGlu is necessary in the formation of the proper conformation of these regulatory peptides for binding to their receptors and/or for protecting the N-termini of these peptides from exopeptidase degradation (Van Coillie et al., Biochemistry 37: 12672-12680 (1998); Hinke et al., J. Biol. Chem. 275: 3827-3834 (2000)). The N-terminal pyroglutamate (pGlu) is formed by the N-terminal cyclization of its glutaminyl precursor. And the glutaminyl cyclases (QCs) are the catalysts responsible for this posttranslational modification (Fischer et al., Proc. Natl. Acad. Sci. USA 84: 3628-3632 (1987); Busby et al., J. Biol. Chem. 262: 8532-8536 (1987)).
QCs (EC 2.3.2.5) are acyltransferases identified in both animal and plant sources (Fischer et al., Proc. Natl. Acad. Sci. USA 84: 3628-3632 (1987); Busby et al., J. Biol. Chem. 262: 8532-8536 (1987); Oberg et al., Eur. J. Biochem. 258: 214-222 (1998)). QCs are abundant in mammalian neuroendocrine tissues, such as hypothalamus and pituitary (Busby et al., J. Biol. Chem. 262: 8532-8536 (1987); Sykes et al., FEBS Lett. 455: 159-161 (1999)), and are highly conserved from yeast to human. Animal QCs were shown to have distinct structure and protein stability from plant QCs in spite of their similar molecular masses, i.e., 33-40 kDa (Oberg et al., Eur. J. Biochem. 258: 214-222 (1998); Schilling et al., Biochemistry 41: 10849-10857 (2002)). While no bacterial QCs have been reported thus far, the mammalian QCs had been predicted to exhibit remarkable homology to the bacterial double-zinc aminopeptidases (Schilling et al., J. Biol. chem. 278: 49773-49779 (2003); Booth et al., BMC biol. 2: 2 (2004)).
Several of human genetic diseases, e.g., osteoporosis that is a multifactorial hormonal disease characterized by reduced bone mass and microarchitectual deterioration of bone tissue (Stewart et al., J. Endocrinol. 166: 235-245 (2000)), appear to result from mutations of the QC gene. The gene encoding QC (QPC7) lies on chromosome 2p22.3. Within the region, thirteen single nucleotide polymorphisms (SNPs) were analyzed and shown a striking correlation with osteoporosis susceptibility in adult women (Ezura et al., J. Bone Miner. Res. 19: 1296-1301 (2004)). Of these SNPs, the R54W presents, statistically, the most prominent association with osteoporosis, which was proposed to affect the pathogenesis through the hypothalamus-pituitary-gonadal axis (Ezura et al., J. Bone Miner. Res. 19: 1296-1301 (2004)).
Interestingly, QC also catalyzes the N-terminal glutamate cyclization that leads to the formation pGlu (Schilling et al., FEBS Lett. 563: 191-196 (2004)). This reaction is probably related to the formation of several plaque-forming peptides, such as amyloid-β (Aβ) peptides and CLAC (collagen-like Alzheimer amyloid plaque component), which play a pivotal role in Alzheimer's disease (Morgan et al., Prog. Neurobiol. 74: 323-349 (2004); Hashimoto et al., EMBO J. 21: 1524-1534 (2002)). Peptides containing N-terminal pGlu, e.g., pGlu3-Aβ peptides, are major fractions of the Aβ peptides within the core of neuritic plaques (Saido et al., Neuron 14: 457-466 (1995); Kuo et al., Biochem. Biophys. Res. Commun. 237: 188-191 (1997); Russo et al., J. Neurochem. 82: 1480-1489 (2002)). The N-terminal pGlu could enhance the hydrophobicity, proteolytic stability and neurotoxicity of these peptides (Russo et al., J. Neurochem. 82: 1480-1489 (2002); Harigaya et al., Biochem. Biophys. Res. Commun. 276: 422-427 (2000)), probably causing a profused accumulation of pGlu-Aβ peptides in several senile plaques, and thus accelerating the progression of neurodegenerative disorders.
To date, there remain several theories concerning the properties and structures of human and animal QCs. The present invention offers the crystal structure of QC in free form, the structures of the active sites or catalytic centers of the QC, the method for identifying an inhibitor of glutaminyl cyclase (QC), and provides a structural basis for the rational design of new inhibitors against QC-associated disorders.